An abnormal thrombin, or dysthrombin, has been purified after fragmentation of Prothrombin Quick, a unique prothrombin variant from a patient with dysprothrombinemia. The goal of the research will be to study the enzymology of the dysthrombin, with a primary objective to determine whether the defect is in substrate binding, catalysis, or both. The activity of the dysthrombin with both natural and synthetic substrates will be determined (specific activity with fibrinogen is less than 1% of that of normal thrombin). Natural substrates will include prothrombin, antithrombin III factors V and VIII and platelets, and synthetic substrates will include arginine esters and tripeptide-p-nitroanilides. Substrates which are hydrolyzed will be analyzed by steady-state kinetics to determine Km and Kcat. Reactivity of the active site serine will be tested by measuring the rate and extent of alkylation of the serine with diisopropylfluorophosphate. The affinity of the dysthrombin for substrates which are not hydrolyzed will be measured directly by spectral titration methods. Abnormalities in function will be related to changes in the primary structure of the dysthrombin which will be analyzed at the Mayo Clinic, Rochester, Minn., under the direction of Dr. Kenneth G. Mann.